Chiral copper(I)—thiolate clusters in metallothionein and glutathione

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal–thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M₇S₂₀ structure are M₄(S_cys)₁₁ (α domain) and M₃(S_cys)₉ (β domain). Up to 12 Cu(I) ions can displace the 7 Zn²⁺ ions bound per molecule in Zn₇–MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu₁₂S₂₀ structure of Cu₆(S_cys)₁₁ and Cu₆(S_cys)₉ for the α and β domains, respectively. The circular dichroism (CD) spectra recorded as Cu⁺ is added to Zn₇–MT to form Cu₁₂–MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(I); because the Cu(I)–thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)–MT species. Model Cu(I)–thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)–thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)–thiolate clusters in the α and β domains of Cu₁₂–MT are proposed. © 1994 Wiley-Liss, Inc.     

Design of a bubble-swawm bioreactor for animal cell culture

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22·10^–3–1.45·10^–3 vvm (30–200 ml/h).The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.     

Genetics and biochemistry of phenol degradation byPseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.     

Transfer and expression of PCB-degradative genes into heavy metal resistantAlcaligenes eutrophus strains

Sites polluted with organic compounds frequently contain inorganic pollutants such as heavy metals. The latter might inhibit the biodegradation of the organics and impair bioremediation. Chromosomally located polychlorinated biphenyl (PCB) catabolic genes ofAlcaligenes eutrophus A5,Achromobacter sp. LBS1C1 andAlcaligenes denitrificans JB1 were introduced into the heavy metal resistantAlcaligenes eutrophus strain CH34 and related strains by means of natural conjugation. Mobile elements containing the PCB catabolic genes were transferred fromA. eutrophus A5 andAchromobacter sp. LB51C1 intoA. eutrophus CH34 after transposition onto their endogenous IncP plasmids pSS50 and pSS60, respectively. The PCB catabolic genes ofA. denitrificans JB1 were transferred intoA. eutrophus CH34 by means of RP4::Mu3A mediated prime plasmid formation. TheA. eutrophus CH34 transconjugant strains expressed both catabolic and metal resistance markers. Such constructs may be useful for the decontamination of sites polluted by both organics and heavy metals.     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.     

Changes in dormancy of Sisymbrium officinaleseeds do not depend on changes in respiratory activity

Effects of dark incubation at different temperatures were studied on dormancy and respiratory activity of seeds of Sisymbrium officinale (L.) Scop. Because germination of this species absolutely depends on the simultaneous action of light and nitrate, changes in dormancy could be studied in darkness without the interference of early germination events. Upon the start of incubation rates of O₂ uptake and CO₂ release rose. This was followed by a gradual decrease until stable levels of O₂ uptake and CO₂ release were achieved. Seeds kept for prolonged periods at 24^°C, showed neither a change in germination capacity nor in rates of O₂ uptake and CO₂ release. Respiratory quotients were 0.55–0.7. The initial rise in O₂ uptake correlated with the rate of water uptake and with breaking of primary dormancy. However, the subsequent decline in O₂ uptake was not generally linked to induction of secondary dormancy. An increased O₂ uptake was not required during breaking of secondary dormancy. It is concluded that changes in dormancy are not generally related to changes in respiratory activity. However, germination strongly depends on respiration. The increase in O₂ uptake started well before radicle protrusion. A far red irradiation only reversed this increase when it was given before germination escaped from its red light antagonising action. The contribution of different respiratory pathways was followed during prolonged incubation at 24^°C in darkness. KCN at 1.5 mM was needed to inhibit the cytochrome pathway (CP) and benzohydroxamic acid (BHAM) at 30 mM to inhibit the alternative pathway (AP). These concentrations did not exert any side effects. Electron flow was predominantly via the CP, maximally 10% was via the AP. Flow through the CP declined during the first 6 days and residual respiration remained constant. Therefore, the contribution of residual respiration became relatively more important with prolonged incubation. KCN at concentrations that almost completely inhibited flow through the CP, did not dramatically reduce germination. BHAM already inhibited germination at concentrations that do not inhibit oxygen uptake.     

P-31 NMR saturation transfer experiments in Chlamydomonas reinhardtii : evidence for the NMR visibility of chloroplastidic Pi

ATP synthesis and consumption in respiring cells of the green alga Chlamydomonas reinhardtii were measured with ³¹P in vivo NMR saturation transfer experiments to determine the intracellular compartmentation of inorganic phosphate. Most of the observed flux towards ATP synthesis was catalyzed by the coupled enzymes glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The attribution of the measured flux to these enzymes is supported by the observation, that (i) the magnetization transfer was strongly reduced by iodoacetate, an irreversible inhibitor of GAPDH and that (ii) the unidirectional flux was much greater than the net flux through the mitochondrial F₀F₁-ATPase as determined by oxygen consumption measurements. In Chlamydomonas, glycolysis is divided into a chloroplastidic and a cytosolic part with the enzymes GAPDH/PGK being located in the chloroplast stroma (Klein 1986). The ³¹P-NMR signal of inorganic phosphate must, therefore, originate from the chloroplast. The life time of the magnetic label transferred to P_i by these enzymes is too short for it to be transported to the cytosol via the phosphate translocator of the chloroplast envelope. When the intracellular compartmentation of P_i was taken into consideration the calculated unidirectional ATP synthesis rate was equal to the consumption rate, indicating operation of GAPDH/PGK near equilibrium. The assignment of most of the intracellular P_i to the chloroplast is in contradiction to earlier reports, which attributed the Pi signal to the cytosol. This is of special interest for the use of the chemical shift of the P_i signal as an intracellular pH-marker in plant cells.Abbreviations 3-PGA 3-phosphoglycerate - CW continuous wave - dG6P 2-deoxyglucose-6-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - MO equilibrium z-magnetization - M₀ instantaneous z-magnetization after selective saturation for time t - MDP methylene-diphosphonic acid - PDE phosphodiester - PGK phosphoglycerate kinase - P_i inorganic orthophosphate - polyP polyphosphate - T₁ longitudinal relaxation time - ₁ longitudinal relaxation time with chemical exchange - TCA cycle tricarboxylic acid cycle Correspondence to: A. Mayer     

Nonlinear effect of dynamic self-organization in macromolecular systems caused by photocontrolled electron flux

We use the electron-conformational interaction approach to develop a physical model which self-consistently describes the photomobilized electron transfer kinetics and structure conformational transitions in reaction centers (RCs) of purple bacteria. We consider the kinetics of electron transition from pigment onto primary acceptor and the subsequent charge recombination accounting for the change of distance between the above-mentioned cofactors. It is shown that, given natural values of RC parameters, the kinetic constant's dependence on the acting light intensity is monotone. As opposed to the previous case, similar dependencies for the chain of electron transfer between primary and secondary quinone acceptors revealed anS-like relationship. This can lead to bistability of the RC optical transmission coefficient and a fundamental dependence of charge recombination kinetics upon the prehistory of the RC's interaction with exciting radiation.     

The sequence of electron carriers in the reaction of cytochromec oxidase with oxygen

Kinetic studies of the electron transfer processes performed by cytochrome oxidase have assigned rates of electron transfer between the metal centers involved in the oxidation of ferrocytochromec by molecular oxygen. Transient-state studies of the reaction with oxygen have led to the proposal of a sequence of carriers from cytochromec, to Cu_A, to cytochromea, and then to the binuclear (i.e., cytochromea ₃-Cu_B) center. Electron exchange rates between these centers agree with relative center-to-center distances as follows; cytochromec to Cu_A 5–7 Å, cytochromec to cytochromea 20–25 Å, Cu_A to cytochromea 14–16 Å and cytochromea to cytochrome a₃-Cu_B 8–10 Å. It is proposed that the step from cytochromea to the binuclear center is the key control point in the reaction and that this step is one of the major points of energy transduction in the reaction cycle.     

Interaction of the FAFB-containing subunit with the Photosystem 1 core heterodimer

The structure of the predicted amino acid sequence in the F_X domain of Photosystem 1 was studied by molecular modeling and a working hypothesis was developed for the functional interaction of PsaC with the core heterodimer. We propose that the intervening sequences between homologous cysteines in the F_X cluster form two flexible loops and participate in the binding of PsaC, and that the arginine residues in the two surface-exposed loops may promote the interaction between the P₇₀₀–F_X core and the subunit. The model was tested experimentally; chemical modification of arginine residues in the P₇₀₀–F_X core using phenylglyoxal prevented reconstitution of the core with PsaC and PsaD after insertion of FeS clusters in vitro. Treatment of the P₇₀₀–F_X core with trypsin also prevented reconstitution of terminal electron transfer to F_AF_B, although neither treatments affected the electron transfer to F_X as judged by flash kinetic spectrophotometry. Electron transfer in the P₇₀₀–F_AF_B complex was not impaired by either phenylglyoxal or trypsin treatment indicating that the small subunit(s) protect the arginine residues that become chemically modified or cleaved. The data are consistent with the working model and point to additional experiments designed to identify the specific residues involved in the interaction between the P₇₀₀–F_X core and PsaC.Abbreviations PG- phenylglyoxal - PS 1- Photosystem 1